Evan A. Talib

Date of Award

Fall 2022

Document Type

Open Access Dissertation

Department

Chemistry and Biochemistry

First Advisor

Caryn E. Outten

Abstract

Iron is an indispensable protein cofactor that plays a critical role in biological functions such as cellular respiration. However, excess iron causes cellular damage via the generation of reactive oxygen species. To obtain optimal intracellular iron levels, eukaryotic cells have developed mechanisms to tightly regulate iron levels. The molecular mechanism for iron regulation in the pathogenic yeast Candida glabrata has not yet been explored, although this yeast bears iron regulation pathways that are very similar to the model yeast Saccharomyces cerevisiae. Understanding how C. glabrata regulates iron levels is critical for the treatment of infections caused by this opportunistic pathogenic yeast. In S. cerevisiae, the transcription factor Aft1 activates the expression of iron uptake genes during iron deficiency, and is deactivated during iron sufficiency by the Fe-S cluster trafficking protein Grx3/4 and Bol2. In order to test whether a similar mechanism controls iron levels in C. glabrata we purified and characterized the homologous proteins Aft1, Grx4, and Bol2 from this yeast pathogen. Our results demonstrate that C. glabrata Grx4 alone and Grx4 in complex with Bol2 can ligate linear [3Fe-4S]+ or [2Fe-2S]2+ clusters following in vitro reconstitution. Circular dichroism spectroscopy demonstrates that the interaction of the [2Fe-2S]2+ cluster-bound Grx4-Bol2 complex with Aft1 is similar to our previous findings from S. cerevisiae. Furthermore, fluorescence anisotropy (FA)-based DNA-binding assays and electrophoretic mobility shift assays (EMSA) studies indicate that C. glabrata Aft1 binds tightly to the iron response element of its target DNA at ~ 1:1 stoichiometry, with a binding constant similar to that previously published for S. cerevisiae Aft2. Taken together, these studies suggest that the Fe-S-dependent mechanism for control of Aft1 activity in C. glabrata closely resembles that in S. cerevisiae.

To further understand iron regulation in S. cerevisiae, we used functional genetics and quantitative RT-PCR to test whether S. cerevisiae Aft1 and its homologue Aft2 impact the activity of the high iron-responsive transcriptional activator Yap5. Previous studies suggested that Yap5 and Aft1 interact in vivo; however, our studies revealed that Yap5 transcriptional activity is not dependent on the expression of either Aft1 or Aft2, ruling out a role for Aft1/2 in Fe-S cluster delivery to Yap5. Overall, our studies on iron regulation proteins in C. glabrata and S. cerevisiae provide novel insight into the molecular mechanisms for iron regulation in both pathogenic and non-pathogenic yeasts.

Rights

© 2022, Evan A. Talib

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